Steroidal sapogenins are known to be useful animal growth stimulators. For example, McKeen et al. (U.S. Pat. No. 3,144,337), the disclosure of which is incorporated herein by reference, disclose animal feeds containing selected steroidal sapogenins that elicit a substantially greater growth increase and feed efficiency response in animals, as compared to feeds which do not contain sapogenins. The beneficial effect of steroidal sapogenins as animal growth stimulants is further discussed by W.H. Hale et al., 1961, Proc. Soc. Exp Biol Med. 106, P. 486-489, the disclosure of which is incorporated herein by reference. McKeen et al. also report that when steroidal sapogenins are concurrently administered with estrogenic substances, such as diethylstilbestrol (DES), estradiol, and the like, the animals exhibit growth responses well beyond those which would be expected from the administration of estrogenic substances alone. However, because substances such as DES are suspected to be carcinogens, and are not always approved as animal growth stimulants, there is an increasing need for materials which will allow efficient incorporation of steroidal sapogenins into animal feeds.
Sapogenins are the acid hydrolysis products of saponins, which are amphiphilic, steroidal or triterpenoid glycoside compounds. Crude extracts containing saponins can be obtained from a wide variety of plant materials. The steroidal saponins, and the steroidal sapogenins derived therefrom, possess the growth stimulating characteristics discussed hereinabove, are characterized by a glycosidic moiety, such as a 5- or 6-membered sugar moiety or a chain of sugar moieties, attached to a steroid moiety by means of an ether linkage at the carbon-3 (C-3) position. The structure of such a steroid moiety is exemplified by the structures of the sapogenins smilagenin, hecogenin and tigogenin which are schematically illustrated in FIG. 1. These structures are representations of sapogenins which would result from hydrolysis of the ether linkage between a glycoside moiety and a steroid moiety of a saponin molecule corresponding to each of these sapogenins. Saponins can be hydrolyzed in this way enzymatically or by exposure to acid.
Steroidal sapogenins are also useful as precursors for pharmaceutical steroids, such as cortisone and various hormones. See e.g. Wall et al., J. Biol. Chem. 198, 533-543; and Printy et al., U.S. Pat. No. 3,169,959.
For steroidal sapogenins to find wide use, an efficient, cost effective way of producing them must be made available. The difficulty of economically obtaining large quantities of steroidal sapogenins has apparently limited their use as animal growth stimulators, and as steroidal precursors.
Although crude aqueous plant extracts containing substantial amounts of steroidal saponins are easily obtained and are commercially available, it has been difficult to isolate the desired steroidal sapogenins from these sources. In addition to saponins, these crude extracts usually contain substantial amounts of plant fats and non-saponin carbohydrates. When these crude extracts are hydrolyzed with concentrated mineral acids, a virtually unusable, sticky, gumlike reaction product generally results. It is believed that the fats and free carbohydrates are responsible for forming this material. Once such a reaction product has formed, it is very difficult to efficiently extract the sapogenins therefrom.
Historically, saponins have been separated from fats and non-saponin carbohydrates by extracting crude aqueous plant extracts with butanol. This method is tedious, and can result in an oily gum which is difficult to handle in an efficient manner and which is not a suitable substrate for subsequent acid hydrolysis to produce sapogenins.
Another method of extracting saponins from plant material has been disclosed involving extraction with hot aqueous ethanol or isopropanol (85-95%). See Wall et al., 1952, J. Biol. Chem. 198, 533-543. The fats are subsequently extracted with benzene, and the saponins are then extracted from the aqueous alcohol solution with butanol. Following these steps, the saponins can be acid-hydrolyzed to form sapogenins. The crude product, however, still requires treatment with hot methanol potassium hydroxide to remove phenolic and acidic substances.
Although Printy et al. (U.S. Pat. No. 3,169,959) have disclosed a method for deriving smilagenin from a crude Agave extract without separating the saponin from the fats and non-saponin carbohydrates prior to hydrolysis, the yield was very low. Smilagenin is considered to be a particularly desireable sapogenin, both for use as a pharmaceutical precursor, and as an animal growth stimulator. Printy et al. extracted pulverized plant material with warm water. The extract was clarified and then hydrolyzed with mineral acid. The crude smilagenin precipitate was isolated and further extracted with heptane to give a 1% yield of smilagenin.
Given the usefulness of steroidal sapogenins as animal growth stimulants and steroid precursors, it will be appreciated that a need exists for a readily obtainable source of steroidal saponins which is substantially free of fats and non-saponin carbohydrates, and which is, therefore, easily hydrolyzed to form steroidal sapogenins. It will be further appreciated that a need exists for an efficient method of producing such an intermediate.